Adrenergic receptors are a family of cell membrane receptors that receive neuronal impulses from postganglionic adrenergic fibers from the sympathetic nervous system. There are at least nine sub-types of adrenergic receptors of which at least three sub-types are beta-adrenergic receptors (βARs) (H. G. Dohlman et al., Annu. Rev. Biochem. 60:653-688 (1991); S. B. Liggett et al., In: Catecholamines, Bouloux, ed. W. B. Sounders, London (1993)). BARs are prototypic G-protein coupled receptors (GPCRs) that play an important role in the regulation of cardiovascular and pulmonary function. β2ARs constitute about 25-30% of the total βARs in the human heart, and are the predominant subtype expressed in both vascular and airway smooth muscle. β2ARs are directly associated with a receptor-channel complex containing G proteins, which activates adenylyl cyclase, cAMP-dependent kinase and the counterbalancing phosphatase, PP2A. The assembly of the signaling complex provides a mechanism that ensures specific and rapid signaling by this G protein-coupled receptor.
Accordingly, β2ARs are expressed in many organs in the body and modulate a variety of physiological functions of the body (J. R. Carstairs et al., Am. Rev. Respir. Dis. 132:541-547 (1985); Q. A. Hamid et al., and Eur. J. Pharmacol. 206:133-138 (1991)). Overexpression of the β2AR greatly increases cardiac contractility and provides a cardioprotective effect (S. B. Liggett et al., Circulation 101, 1707 (2000)). Continuous agonist stimulation induces β2AR ubiquitylation, internalization and degradation (S. K. Shenoy, P. H. McDonald, T. A. Kahout, R. J. Leftkowitz, Science 294, 1307 (2001)), thereby down-regulating the total number of receptors. βAR down-regulation (and dysfunction) is associated with diseases such as heart failure and asthma (H. A. Rodman, W. J. Koch, R. J. Leftkowitz 425, 206 (2002); M. Johnson An J Respir. Crit. Care Med. 158, S146 (1998)).
Given the importance of β2ARs in modulating physiological functions, there is a need in the art for regulating the expression and activity of β2ARs.
The inventors of the present application have discovered that the Von Hippel-Lindau tumor suppressor protein (pVHL)-E3 ligase complex, characterized previously by its regulation of hypoxic inducible factor (HIF) proteins, interacts with and ubiquitylates β2AR, promoting receptor down-regulation. Under normoxic conditions HIF proteins are very unstable due to hydroxylation by a family of proline hydroxylases termed EGL-nine homologs (EGLN).
The interaction of pVHL with the β2AR is dependent on proline hydroxylation, and the dioxygenase EGLN3 interacts directly with the β2AR to serve as an endogenous β2AR prolyl hydroxylase. Following hydroxylation of the β2AR, the pVHL-E3 ligase complex is recruited to and ubiquitylates the β2AR, promoting its down-regulation via proteosomal degradation.
The inventors of the present application have further discovered that the interaction between EGLN3 and β2AR can be manipulated to regulate the expression and activity of β2ARs.